Molecular Mechanism Of Activation Of Macrophage Inhibited By Peaoniflorin According To JAK2/STAT3signal Transduction In Diabetic Kidney

Background and purpose:Diabetic nephropathy(DN)is still one of the main causes of end-stage renal failure(ESRF).The pathogenesis of DN and its treatment have always been one of the focuses of researchers.In recent years,there has been much evidence that intrarenal infiltration and activation of macrophages cause the release of associated inflammatory factors,such as NF-κB activation,which increases the levels of inflammatory factors.This accelerates the development of DN.However,the signal transduction pathways involved in the activation of macrophages in diabetic renal tissues are still not elucidated.A large number of studies have shown that monocytes/macrophages that participate in inflammatory responses first require activation of the JAK2/STAT3 pathway to activate downstream signaling pathways in order to recruit and activate macrophages,initiate a series of inflammatory responses,and persistently aggravate kidney damage in people with diabetes.It is hypothesized that this is crucial to the development of microvascular complications in diabetics and a beneficial target of anti-inflammatory treatment.In recent years,the use of traditional Chinese medicine in DN treatment has attracted attention.Paeoniflorin(PF)is an active ingredient of total glucosides of peony and has pharmacological effects such as anti-inflammatory activation,and anti-oxidant and immune activity.In this study,PF was used in a streptozotocin(STZ)-induced diabetic model and high glucose-stimulated RAW 264.7 mouse macrophage cell lines to observe changes in the JAK2/STAT3 signaling pathway and pro-inflammatory factors from an overall,cellular,and molecular levels.These experiments are used to observe the mechanisms by which the JAK2/STAT3 signaling pathway participates in DN occurrence and PF intervention,in order to provide new ideas for the prevention and treatment of DN.Methods:Part 1:The RAW264.7 macrophage cell line was used.The CCK-8 assay was employed to detect the effects of PF intervention and high glucose stimulation on cell activity of RAW 264.7 macrophages.The chemotaxis experiment was used to observe the effects of high glucose stimulation,PF intervention,and JAK2/STAT3 gene silencing on the chemotactic functions of RAW 264.7 macrophages.Different concentration gradients of high glucose were used to stimulate cells,and different concentration gradients of PF were used for intervention to observe the effects of different glucose concentrations on the expression of signaling pathway proteins in RAW 264.7 macrophages at different time points,thereby determining suitable concentrations for glucose and PF needed for the experiment and specific time points for experimental observations.The aforementioned results were used to divide RAW264.7 macrophages into groups,namely the normal glucose concentration(LG)group,high glucose stimulation(HG)group,high glucose+paeoniflorin(HG+PF)group,JAK2 gene silencing(JAK2 siRNA)group,JAK2 gene silencing+high glucose(JAK2 siRNA+HG group),JAK2 gene silencing+high glucose+paeoniflorin(JAK2siRNA+HG+PF group),STAT3 gene silencing(STAT3 siRNA)group,STAT3 gene silencing+high glucose(JAK2 siRNA+HG group),and STAT3 gene silencing+high glucose+paeoniflorin(JAK2 siRNA+HG+PF group).Cells and cell culture supernatants were collected at corresponding simulation timepoints.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the expression of the pro-inflammatory factors,TNF-α,MCP-1,and IL-1βin cell culture supernatant.Real-time quantitative polymerase chain reaction(PCR)was used to measure the mRNA expression of various pro-inflammatory cytokines,such as TNF-α,MCP-1,IL-1β,and iNOS.Western blotting was used to measure the protein expression of p-JAK2,p-STAT2,and iNOS in various RAW 264.7 macrophage groups.Part 2:Healthy,male wild-type C57BL/6J mice from the same litter were given 50mg/kg of STX by intraperitoneal injection for 5 days.One week after injection,random blood glucose was measured from the tail veins of mice.Mice with blood glucose≥16.7mmol/L were confirmed to be diabetic mouse models.Model mice were randomized into the diabetic model group,PF administration(25,50,and 100 mg/kg/d)group and the control group,with 12 mice per group.At the end of week 12 after drug administration,blood,urine,and kidney tissue samples from the different groups were collected.Blood glucose,body weight,kidney weight,and 24-hour urinary albumin excretion were measured,and renal histopathological changes were observed.Immunohistochemistry was used to quantitate CD68,p-JAK2,and p-STAT3 protein expression.Western blotting was used to quantitate the expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 in kidney tissues.Real-time quantitative PCR was used to quantitate the mRNA expression levels of TNF-α,MCP-1,IL-1β,and iNOS.ResultsPart 1:(1)Compared with the normal controls,the PF concentrations used in the experiments did not have any significant effects on cell activity of RAW 264.7macrophages in a high glucose environment(P>0.05).(2)Western blot results showed that elevation in p-JAK2 expression was the most significant at 25 mmol/L(P<0.01)and elevation in p-STAT3 expression was the most significant at 25 mmol/L glucose(P<0.01).p-JAK2 reached its peak(P<0.01)at 15 minutes of stimulation while p-STAT3 reached its peak at 24 hours of stimulation(P<0.01).Therefore,we selected a glucose concentration of 25 mmol/L for subsequent culture and a stimulation time of24 hours was selected as the endpoint for the experiment.Compared with the LG group,p-JAK2 and p-STAT3 protein expression were all elevated(P<0.05).Compared with the HG group,when PF concentration was 10~-66 mmol/L,p-JAK2 and p-STAT3protein expression in RAW 264.7 macrophages could be decreased and 10~-55 mmol/L of PF had the strongest inhibitory effect on p-STAT3 protein expression(P<0.01).Therefore,we selected 10~-55 mmol/L of PF as the intervention concentration for subsequent experiments.(3)Chemotaxis experiments showed that high glucose stimulation could increase macrophage chemotaxis by MCP-1,resulting in macrophage migration and PF intervention can inhibit the stimulatory effects of high glucose.(4)Real-time fluorescence quantitative PCR results showed that compared with the LG group,iNOS,TNF-α,IL-1β,and MCP-1 mRNA levels in the HG group were significantly upregulated(P<0.01).Compared with the LG group,iNOS,TNF-α,IL-1β,and MCP-1 mRNA levels showed varying degrees of decrease in the JAK2 siRNA and STAT3 siRNA groups(P<0.01).Compared with the HG group,STAT-3 siRNA and PT intervention could decrease iNOS,TNF-α,IL-1Β,and MCP-1mRNA levels to varying degrees(P<0.01).Compared with the JAK2 siRNA/STAT3siRNA groups,the PF group showed further downregulation of iNOS,TNF-α,IL-1β,and MCP-1 mRNA levels(P<0.01).(5)The ELISA results showed that compared with the LG group,levels of secreted TNF-α,IL-1β,and MCP-1 in the cell culture medium of the HG group were significantly upregulated(P<0.01).Compared with the LG group,JAK2 siRNA and STAT siRNA could reduce the levels of secreted TNF-α,IL-1β,and MCP-1 to varying degrees(P<0.05,P<0.01).Compared with the HG group,JAK2 siRNA,STAT-3 siRNA,and PF intervention could decrease the levels of secreted TNF-α,IL-1β,and MCP-1(P<0.01).Compared with the JAK2 siRNA/STAT3 siRNA groups,PF intervention could further decrease the levels of secreted TNF-α,IL-1β,and MCP-1(P<0.01).(6)Laser confocal microscopy observations showed that the fluorescence intensity of the M1 macrophage-specific marker,iNOS,was significantly higher in the HG group than the LG group while JAK2/STAT3 gene silencing could significantly reduce iNOS fluorescence intensity.JAK2/STAT3 gene silencing and PF intervention could reduce iNOS fluorescence intensity under glucose stimulation while PF intervention could further reduce iNOS fluorescence intensity after JAK2/STAT3 gene silencing.Part 2:(1)At week 12,the blood glucose,kidney/body weight,and 24-hour urinary albumin excretion rate of the diabetic model group was significantly increased(P<0.01)compared with the control group:changes in body weight,kidney weight,and 24-hour urinary albumin excretion rate in mice from the PF intervention group were all significantly lower than the diabetic model group(P<0.05,P<0.01).(2)At week 12,optical microscopy showed increased glomerular volume,increased numbers of mesangial cells,thickening of the basement membrane,and increased extracellular matrix in the kidney tissues from the diabetic model group.Under PF intervention,pathological changes in kidney tissues were significantly alleviated(P<0.05,P<0.01).(3)Immunohistochemistry results showed that at week 12,compared with the control group,CD68 expression in diabetic model mice was significantly upregulated(P<0.01):compared with the PF intervention group,CD68 expression was significantly downregulated(P<0.01).The expression of the p-JAK2 protein in the glomerulus and the renal tubulointerstitial cells in the model group were significantly higher than the control group(P<0.01).However,only the p-STAT3 protein in the renal tubulointerstitial cells in the model group was significantly higher than the control group(P<0.01).PF administration significantly inhibited p-JAK2 protein expression in the glomerulus and the renal tubulointerstitial cells(P<0.05:P<0.01)and p-STAT3 protein expression in the renal tubulointerstitial cells were also significantly inhibited(P<0.05,P<0.01).(4)Analysis of band optical density in Western blot results showed that the expression of p-JAK2/JAK2 and p-STAT3/STAT3 in mice in the model group were significantly increased compared with the control group(P<0.01).Administration of 25,50,and 100 mg/kg/d PF for 12 weeks could significantly decrease p-JAK2/JAK2 and p-STAT3/STAT3 expression in kidney tissues(P<0.01).Real-time quantitative PCR results showed that compared with the control group,the mRNA expression of pro-inflammatory factors(TNF-α,MCP-1,IL-1β,and iNOS)in the diabetic model mice was significantly increased(P<0.01).When compared with the model group,the mRNA expression of TNF-α,MCP-1,IL-1β,and iNOS in the PF intervention group was significantly inhibited(P<0.01).Conclusions:1)Under a high glucose environment,RAW 264.7 macrophages are activated,and there is high iNOS expression and upregulation of intracellular JAK2/STAT3 and downstream signaling pathways.This increases the expression of pro-inflammatory factors.PF intervention and JAK2/STAT3 gene silencing can inhibit the activation of these aforementioned signaling pathways,downregulate the expression of macrophage inflammatory factors,and inhibit phenotypic changes in macrophages at the same time.2)Studies have shown that the expression of this protein in diabetic kidney tissue has a certain increase,while macrophage infiltration,inflammatory reaction-related lesions.The signal pathway also plays a role in judging the occurrence of the disease.3)STZ induced activation and increased expression of inflammatory factors and nitric oxide synthase.The expression level of these proteins decreased under the influence of paeoniflorin.This signaling pathway is also closely related to the occurrence of diabetic nephropathy.PF has a certain blocking effect on the activation of this signaling pathway,and slows down the related inflammatory response,thus has a certain therapeutic effect on diabetic nephropathy,but its related mechanism is not clear,which needs further study.